In a chimeric molecule, two or more molecules that exist separately in their native state are joined together to form a single molecule having the desired functionality of all of its constituent molecules. Frequently, one of the constituent molecules of a chimeric molecule is a “targeting molecule”. The targeting molecule is a molecule such as an antibody that specifically binds to its corresponding target and, by virtue of the targeting molecule, the chimeric molecule will specifically bind (target) cells and tissues bearing the target (e.g. the epitope) to which the targeting moiety is directed.
Another constituent of the chimeric molecule may be an “effector molecule”. The effector molecule refers to a molecule that is to be specifically transported to the target to which the chimeric molecule is specifically directed.
Chimeric molecules comprising a targeting moiety attached to an effector moiety have been used in a wide variety of contexts. Thus, for example, chimeric molecules comprising a targeting moiety joined to a cytotoxic “effector molecule” have frequently been used to target and kill tumor cells (see, e.g., Pastan et al., Ann. Rev. Biochem., 61: 331-354 (1992). Other chimeric molecules comprising a targeting moiety attached to angiogenesis inhibitors have been used to inhibit tumor growth and/or proliferation. Conversely, angiogenesis inducers) have been proposed for the treatment of atherosclerosis. Other uses of chimeric molecules have involved the delivery of intrabodies, intracellularly expressed antibodies that then bind to an intracellular protein, the specific delivery of vectors (e.g. for gene therapy), or the creation of tissue-specific liposomes.
Typically, the target recognized by the targeting moiety is not the desired site of action of the effector molecule. Thus, for example, in the case of chimeric cytotoxins used to treat cancers (e.g. IL4-PE, B1FvPE38, etc., see, e.g., Benhar & Pastan (1995) Clin. Canc. Res., 1: 1023-1029, Thrush et al. (1996) Ann. Rev. Immunol., 14: 49-71, etc.) the targeting moiety specifically binds to a target on the surface of the cell. The chimeric molecule is then internalized into the cell and the effector molecule (e.g., ricin, abrin, Diptheria toxin, Pseudomonas exotoxin) is transported to the cytosol of the cell where it exerts its characteristic activity (e.g. ADP ribosylation in the case of Pseudomonas exotoxin).
Similarly, targeted liposomes are typically internalized through a receptor-mediated process or through the action of the lipid. Targeted intrabodies and gene therapy vectors are also internalized for expression within the cell. In addition, a common goal in the design of targeted chimeric molecules has been the increase of binding specificity and avidity. It is generally believed that, by increasing avidity and specificity the concentration of the chimeric molecule to achieve a given result will decrease. Thus, release of the chimeric molecule from its target is generally viewed as undesirable.
Because the chimeric molecule is typically internalized (in the case of targeted cells) and the activity of the effector molecule is directed to a molecule other than the specifically recognized target, chimeric molecules typically act in a “stoichiometric” manner. That is, each chimeric molecule is essentially consumed upon interaction with its “substrate” and activity of the chimeric molecule is unavailable for subsequent reactions. As a consequence chimeric molecules must be maintained at relatively high level for efficacy and a recurring problem of chimeric moieties, particularly in in vivo applications is the inability to maintain elevated serum levels of the chimeric molecule over therapeutically significant periods of time and the increased (e.g. non-specific) toxicity caused by the high dosages that must be utilized.
Attempts at solving these problems have focused on reducing the immunogenicity of the chimera (e.g. by using humanized antibodies, antibody fragments, small fusion proteins, etc.) or “masking” the chimeric molecule (e.g. “stealth” liposomes). In particular, the impetus to reduced immunogenicity, improved tumor penetration, and the like, has led to the increasing use of fusion proteins instead of chemically coupled moieties in chimeric molecules (see, e.g., Pastan, (1992) Ann. Rev. Biochem., 61: 331-354; Thrush (1996) Ann. Rev. Immunol., 14: 49-71; Brinkmann and Pastan (1994) Biochim. Biophys. Acta, 1198: 27-45, etc.), but have not addressed the actual stoichiometry or kinetics of the chimera.